Inflammatory processes generally contribute to host defense against infections and as a stress response to tissue injury. Conversely, inflammation contributes to the chronic or acute pathological processes in autoimmune and cardiovascular diseases and other conditions that lead to tissue injury and destruction. Inflammation is a hallmark of several vascular diseases including atherosclerosis, restenosis, and the vasculopathy associated with transplantation. The most common vascular disease, atherosclerosis, begins when lipoproteins, and in particular low density lipoprotein (LDL) enter the subendothelium and become oxidized. Oxidized LDL stimulates the production of interleukin-1 and other inflammatory cytokines. These cytokines activate adhesion molecules, including VCAM-1, ICAM-1, and E-selectin, on the endothelial surface, which promote the attachment of, and transmigration of monocytes. The expression of the inducible form of nitric oxide synthase (NOS2) has also been shown to be upregulated by inflammatory cytokines and endotoxin in cultured cells found in the atherosclerotic plaque including macrophages, smooth muscle cells, T lymphocytes, and endothelial cells (Esaki, T., et al. 1997. Atherosclerosis 128:39-46; Rikitake, Y., et al. 1998, Atherosclerosis 136:51-7; Xu, R., et al. 1999, Life Sci 64:2451-62). Furthermore, immunohistological studies have demonstrated the expression of NOS2 in the atherosclerotic lesions in these cell types as well (Buttery, L. D., et al. 1996, Lab Invest 75:77-85; Esaki, T., et al. 1997, Atherosclerosis 128:39-46).
The induction of the NOS2 gene is also associated with more acute forms of vascular inflammation such as endotoxemia. The generation of the potent vasodilator, nitric oxide (NO) by NOS2, is at least in part responsible for the hypotension seen in association with bacterial sepsis (Wei, 1995 #1021) (MacMicking, 1995 #1020). NOS2 gene expression is also induced in other types of vascular inflammation including restenosis and in the accelerated atherosclerosis associated with heart transplantation (Ikeda, U., et al. 1998, Clin Cardiol 21:473-6; Lafond-Walker, A., et al. 1997, Am J Pathol 151:919-25).
Rheumatoid arthritis (RA) is a prototypical immune-mediated disease characterized by chronic inflammation in the synovium and the destruction of joints, in which, similar to other inflammatory disorders, a central role for interleukin (IL)-1 and tumor necrosis factor (TNF)-α has been established. These cytokines and bacterial endotoxins have major roles in inflammatory responses via the activation of a variety of transcription factors.
Upon binding of cytokines or other inflammatory mediators to their corresponding receptors, several classes of transcription factors function as mediators of these stimuli. For example, within minutes of interleukin-1 beta (IL-1β treatment, the expression of the immediate early genes cFos and c-Jun are induced. These transcription factors are the constituent proteins for AP-1 (Conca, W., et al. 1991, J Biol Chem 266:16265-8; Conca, W., et al. 1989, J Clin Invest 83:1753-7). One of the target genes of IL-1β, the collagenase gene, can be activated by AP-1 alone (Angel, P., I. et al. 1987, Mol Cell Biol 7:2256-66). Multiple signaling pathways have been implicated in the activation of these immediate early genes by IL-1β including the Janus kinases (JAKs), MAP kinases, and protein kinase A (Hill, C. S., and R. Treisman. 1995, Cell 80:199-211; Karin, M. 1995, J Biol Chem 270:16483-6; Sadowski, H. B., et al. 1993, Science 261:1739-44; Treisman, R. 1996, Curr Opin Cell Biol 8:205-15; Wagner, B. J., et al. 1990, Embo J 9:4477-84).
The propagation of inflammation is dependent on several other transcription factors for the activation of multiple genes. The nuclear factor kappa B (NF-κB) transcription factors are dimeric proteins involved in the activation of a large number of genes in response to inflammatory stimuli. Although originally described to have been important in lymphoid cells and lymphoid specific genes, NF-κB has clearly been shown to play an important role in a whole host of other cell types and target genes. The p50 and p65 subunits of NF-κB have also been shown to bind to other transcription factors through protein interactions often resulting in synergistic transactivation of the target genes of NF-κB (Baeuerle, P. A., and D. Baltimore. 1996, Cell 87:13-20; DiDonato, J. A., et al. 1997, Nature 388:548-54).
One of the major transcriptional circuits implicated in inflammation is the NF-κB/IκB pathway. NF-κB is rapidly activated by proinflammatory cytokines and endotoxins and is involved in the regulation of a large set of inflammatory response genes including various cytokines and chemokines, acute phase proteins, cell adhesion proteins, immunoglobulins, and viral genes. Most of these genes are directly regulated by NF-κB via high affinity binding sites within their respective promoter regions. However, the regulation of a significant number of inflammatory response genes by cytokines cannot be attributed exclusively to direct interaction of NF-κB with binding sites within their regulatory regions, thereby suggesting that additional pathways play critical roles in the transcriptional regulation of these genes.
Osteoarthritis (OA) is another example of a disease having an inflammatory response. OA is a slowly progressive disease with multiple etiologies involving biomechanical, biochemical, and genetic factors, all of which may contribute to the OA lesion in cartilage by disrupting chondrocyte-matrix associations and altering metabolic responses in the chondrocyte. The central role of cytokines, particularly interleukin (IL)-1 and tumor necrosis factor (TNF)-α, in causing the destruction of articular cartilage is well established. It is generally accepted that the chondrocyte is the target of cytoline action, although the sources responsible for generating the cytokines are less clear in the context of OA pathogenesis. Even in the absence of classical inflammation characterized by infiltration of neutrophils and macrophages into joint tissues, elevated levels of proinflammatory cytokines have been measured in OA synovial fluids. Nevertheless, symptoms of local inflammation and synovitis are present in many patients and in animal models of OA. Thus, the fibroblast- and macrophage-like synovial cells, as well as the chondrocytes themselves, are potential sources of cytokines that could induce chondrocytes to synthesize and secrete cartilage-degrading proteases and other cytokines and proinflammatory mediators.
The complexity of the cytokine network that may be involved in OA has increased with the recent discoveries of additional proinflammatory and destructive, as well as inhibitory, cytokines that may amplify or modify the effects of the primary catabolic cytokines. Changes in the patterns of the production of growth factors or their receptors may also contribute to the course of the disease. Aspects of the role of the chondrocyte in OA and lessons from animal models have been reviewed recently. (Goldring M B, Connec Tiss Res 1999, 40:1-11; Goldring M B. Arthritis Rheum 2000, in press.).
Cytokines and growth factors are produced in joint tissues and released into the synovial fluid, and they act on the resident cells in an autocrine-paracrine manner. Many of these factors are necessary at low levels for normal homeostasis, but in OA their balance may be disturbed. The major proinflammatory cytokines, which are generally also catabolic, include IL-1α and β, TNF-α, IL-6, leukemia inhibitory factor (LIF), oncostatin-M (OSM), IL-8, IL-17, and IL-18. The anti-inflammatory cytokines, IL-4, IL-10, IL-11, IL-13, IL-1 receptor antagonist (IL-1ra) and IFN-γ, are classified as inhibitory cytokines, since they may block the actions of catabolic cytokines. Members of the transforming growth factor (TGF)-β/bone morphogenetic protein (BMP) family, insulin-like growth factor-I, and fibroblast growth factors (FGFs) are considered to be major anabolic factors for cartilage, since they may oppose cartilage destruction by promoting synthesis of matrix proteins. Some of these factors, such as IL-6 and TGF-β, may have dual roles. The role of cytokines in osteoarthritis is described in further detail in Goldring, M. B., Current Rheumatology Reports, Jul. 1, 2000, incorporated by reference in its entirety.
The Ets genes are a family of at least thirty members that function as transcription factors (Wasylyk B., H. S. L., Giovane A. 1993, Eur. J. Biochem. 211:7-18). All Ets factors share a highly conserved 80-90 amino acid long DNA binding domain, the ETS domain. Ets factors play a central role in regulating genes involved in development, cellular differentiation and proliferation. Many macrophage, B and T cell specific genes are regulated by Ets factors. The role of Ets factors in the immune system has been substantiated by experiments in mice where the genes encoding several Ets factors have been disrupted by homologous recombination. The PU.1 knockout is characterized by a lack of immune system development (Scott, E. W., et al. 1994, Science 265:1573-7). The Ets-1 knockout mice are characterized by T cell apoptosis and increased terminal B cell differentiation (Muthusamy, N., K. Barton, and J. M. Leiden. 1995, Nature 377:639-42).
Epithelial cell-specific members of the Ets transcription factor family, i.e., ESE-2, ESE-3, and PDEF have been isolated. Recently, a novel member of the Ets factor, called ESE-1, was discovered. Under normal physiological conditions ESE-1 expression is restricted to many epithelial cell types in a variety of tissues with highest expression in the gastrointestinal tract.
ESE-1 is the prototype member of a new subclass of Ets factors and has several interesting features when compared to other Ets family members. First, unlike other Ets factors which are either ubiquitously expressed or primarily expressed in lymphoid cells, ESE-1 appears to have an epithelial-specific expression pattern under basal conditions. Second, unlike all other Ets factors, ESE-1 has two DNA binding domains, a classical Ets domain and in addition an A/T hook domain also found in high mobility group (HMG) proteins. (See Oettgen, P., et al. 1996, Mol Cell Biol 16:5091-106; Oettgen, P., et al. 1999, Genomics 55:358-62; Oettgen, P., et al. 1997, Genomics 445:456-7; and Oettgen, et al., 1997, Mol. Cell Biol. 17(8):4419-33. These references are incorporated herein in their entirety).
One important medical need is the effective treatment of cardiovascular disease, inflammation, and autoimmune diseases. At the moment these diseases are treated with drugs that have inadequate safety profiles and limited efficacy. Currently, the therapeutic alternatives available to treat inflammation consist of the use of corticosteroids or non-steroidal anti-inflammatory agents (NSAIDS). Unfortunately, all of these anti-inflammatory agents are associated with significant side effects, including gastrointestinal irritation and bleeding, bone loss, and fluid retention, some of which can be life-threatening. These anti-inflammatory drugs are therefore not ideal therapeutic agents. Other drugs that target only a single gene involved in inflammatory processes are not effective enough, since only a single component of inflammation is targeted leaving all the other components untouched.
For example, experimental approaches for OA therapy have targeted production or activities of catabolic cytokines. In addition to anticytokine therapy, selective MMP inhibitors targeting enzymes that degrade cartilage-specific collagens and aggrecan also offer the potential to halt cartilage damage. Protein kinases that regulate signal transduction pathways induced by catabolic cytokines have also been proposed as therapeutic targets (Lewis A J, et al. Curr Opin Chem Biol 1999, 3:489-494; Badger A M, et al. Arthritis Rheum 2000, 43:175-183). These include the stress-activated protein kinases (SAPKs), c-Jun N-terminal kinases (JNKs) and p38 MAP kinase, and the IKK1 and IKK2 kinases that release nuclear factor (NF)-κB from its inhibitor IκB, which are known to be activated in chondrocytes by catabolic cytokines. The loss of cartilage in OA is a consequence not only of the disturbed production of and responsiveness to catabolic factors, but also the failure of cartilage repair once it begins to breakdown. Thus, therapy should begin early and target pivotal catabolic pathways without affecting normal homeostasis. Current procedures for repairing or transplanting articular cartilage that is more severely damaged do not provide long-term restoration of the normal cartilage surface (Buckwalter J A, et al. Arthritis Rheum 1998, 42:1331-1342.). Autologous chondrocyte transplantation has been used somewhat successfully in traumatic defects of knee cartilage in young adults. However, the repair of more extensive defects in OA patients will require the development of approaches for genetically engineering chondrocytes prior to transplantation to not only promote cartilage-specific matrix synthesis, but also to counteract the effects of inflammatory and catabolic cytokines (Evans C H, et al., Arthritis Rheum 1999, 42:1-16.).
Pharmacological interventions for OA have focused primarily on improving symptoms, although new agents may offer the possibility of preserving normal homeostasis. Since the increased synthesis of MMPs, prostaglandins, and other inflammatory and catabolic factors in OA tissues appears to be related to elevated levels of IL-1 and TNF-α, therapies that interfere with the expression or actions of these cytokines are most promising.
It would be useful to have effective methods of treating different types of inflammation, such as vascular inflammatory disorders, rheumatologic disorders, dermatologic inflammatory diseases, gastrointestinal inflammatory diseases and kidney disorders, to name a few. It would be especially useful to have methods of treating inflammation that modulate the transcription factors that mediate inflammation. It would also be useful to have methods of screening compounds that are capable of reducing an inflammatory response, especially methods that modulate the expression of the transcription factors involved in the response.